Friedreich's ataxia: Autosomal recessive disease caused by an intronic GAA triplet repeat expansion

Science

Volumn 271, Issue 5254, 1996, Pages 1423-1427

  Campuzano, Victoria ^a   Montermini, Laura ^b   Moltò, Maria Dolores ^b,f   Pianese, Luigi ^c   Cossée, Mireille ^a   Cavalcanti, Francesca ^c,d   Monros, Eugenia ^e   Rodius, François ^a   Duclos, Franck ^a,j   Monticelli, Antonella ^c   Zara, Federico ^b   Cañizares, Joaquin ^f   Koutnikova, Hana ^a   Bidichandani, Sanjay I ^b   Gellera, Cinzia ^g   Brice, Alexis ^h   Trouillas, Paul ⁱ   De Michele, Giuseppe ^c   Filla, Alessandro ^c   De Frutos, Rosa ^f   Palau, Francisco ^e   Patel, Pragna I ^b   Di Donato, Stefano ^g   Mandel, Jean Louis ^a   Cocozza, Sergio ^c   Koenig, Michel ^a   Pandolfo, Massimo ^b,g   more..

^a INSTITUT DE GÉNÉTIQUE ET DE BIOLOGIE MOLÉCULAIRE ET CELLULAIRE   (France)

^b BAYLOR COLLEGE OF MEDICINE   (United States)

^c UNIVERSITY OF NAPLES FEDERICO II   (Italy)

^d IRCCS NEUROMED   (Italy)

^e HOSPITAL UNIVERSITARIO LA FE   (Spain)

^f UNIVERSITY OF VALENCIA   (Spain)

^g DEPARTMENT OF NEUROSURGERY   (Italy)

^h PITIÉ SALPÊTRIÈRE HOSPITAL   (France)

ⁱ HÔPITAL NEUROLOGIQUE   (France)

^j UNIVERSITY OF IOWA   (United States)

more..

Author keywords

[No Author keywords available]

Indexed keywords

PROTEIN;

ARTICLE; AUTOSOMAL RECESSIVE DISORDER; CAENORHABDITIS ELEGANS; CENTRAL NERVOUS SYSTEM; CHROMOSOME 9Q; CONTROLLED STUDY; DEGENERATIVE DISEASE; FETUS; FRIEDREICH ATAXIA; GENE LOCUS; HEART DISEASE; HUMAN; INTRON; MAJOR CLINICAL STUDY; NUCLEOTIDE SEQUENCE; PERIPHERAL NERVOUS SYSTEM; POINT MUTATION; PRIORITY JOURNAL; YEAST;

EID: 13344270899     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5254.1423     Document Type: Article

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24. +] RNA [J. G Morgan et al., Nucleic Acids Res. 20, 5173 (1992)]. Finally, seven of the cosmids were subcloned as Sau 3AI, Apo I, and Hae III fragments, and ∼1500 random single-pass sequences were generated, which were then analyzed with the GRAIL1a and GRAIL2 [E. C Uberbacher and R. J. Mural, Proc. Natl. Acad. Sci U.S.A. 88, 11261 (1991)] and FEXH [V. V. Solovyev, A A. Salamov, C. B. Lawrence, Nucleic Acids Res. 22, 5156 (1994)] programs These three approaches yielded 19, 5, and 17 potential coding sequences, respectively, including two that matched known genes, namely the protein kinase A γ catalytic subunit gene (obtained by cDNA selection and random sequencing) and a mitochondrial adenylate kinase 3 pseudogene (obtained by random sequencing)
25. +] RNA [J. G Morgan et al., Nucleic Acids Res. 20, 5173 (1992)]. Finally, seven of the cosmids were subcloned as Sau 3AI, Apo I, and Hae III fragments, and ∼1500 random single-pass sequences were generated, which were then analyzed with the GRAIL1a and GRAIL2 [E. C Uberbacher and R. J. Mural, Proc. Natl. Acad. Sci U.S.A. 88, 11261 (1991)] and FEXH [V. V. Solovyev, A A. Salamov, C. B. Lawrence, Nucleic Acids Res. 22, 5156 (1994)] programs These three approaches yielded 19, 5, and 17 potential coding sequences, respectively, including two that matched known genes, namely the protein kinase A γ catalytic subunit gene (obtained by cDNA selection and random sequencing) and a mitochondrial adenylate kinase 3 pseudogene (obtained by random sequencing)
26. +] RNA [J. G Morgan et al., Nucleic Acids Res. 20, 5173 (1992)]. Finally, seven of the cosmids were subcloned as Sau 3AI, Apo I, and Hae III fragments, and ∼1500 random single-pass sequences were generated, which were then analyzed with the GRAIL1a and GRAIL2 [E. C Uberbacher and R. J. Mural, Proc. Natl. Acad. Sci U.S.A. 88, 11261 (1991)] and FEXH [V. V. Solovyev, A A. Salamov, C. B. Lawrence, Nucleic Acids Res. 22, 5156 (1994)] programs These three approaches yielded 19, 5, and 17 potential coding sequences, respectively, including two that matched known genes, namely the protein kinase A γ catalytic subunit gene (obtained by cDNA selection and random sequencing) and a mitochondrial adenylate kinase 3 pseudogene (obtained by random sequencing)
27. +] RNA [J. G Morgan et al., Nucleic Acids Res. 20, 5173 (1992)]. Finally, seven of the cosmids were subcloned as Sau 3AI, Apo I, and Hae III fragments, and ∼1500 random single-pass sequences were generated, which were then analyzed with the GRAIL1a and GRAIL2 [E. C Uberbacher and R. J. Mural, Proc. Natl. Acad. Sci U.S.A. 88, 11261 (1991)] and FEXH [V. V. Solovyev, A A. Salamov, C. B. Lawrence, Nucleic Acids Res. 22, 5156 (1994)] programs These three approaches yielded 19, 5, and 17 potential coding sequences, respectively, including two that matched known genes, namely the protein kinase A γ catalytic subunit gene (obtained by cDNA selection and random sequencing) and a mitochondrial adenylate kinase 3 pseudogene (obtained by random sequencing)
28. The 5′-RACE experiment was performed with the Clontech RACE-Ready cDNA kit, according to the manufacturer's instruction
29. The EST (GenBank accession number R06470) represent 5′ sequence of cDNA clone 126314 determined by the Washington University-Merck EST Project.
30. The EST clone contained four exons, and the longer RACE product contained one additional 5′ exon This exon mapped within the CpG island at position 100 on the genomic map. A transcription start site was predicted 388 bp upstream of the exon 1 donor splice site, and a TATA box could be found 28 bp further upstream by the TSSG program [V. V. Solovyev, A. A. Salamov, C. B. Lawrence, in preparation].
31. An in-frame ATG preceded 108 bp upstream by an in-frame stop codon was found in exon 1, and it was assumed to be the translation start site.
32. The 3′-RACE experiment was carried out as described [E. M. Frohman and J. B. Martin, Technique 1, 165 (1989)], with total RNA from HeLa cells (2 mg) and nested primers in exon 5b.
33. This longer 3′-RACE product ended with the poly(A) tail of a downstream Alu sequence. The genomic sequence of exon 6 showed that it contains three Alu sequences in tandem, followed by a poly(A) signal 1050 bp away from the acceptor splice site Exon 6 was mapped 13 kb telomeric to exon 5b (Fig. 1A). Splice sites of all seven exons (1 to 4, 5a, 5b, and 6) conform to the canonical consensus.
34. Further hybridizations of the Northern blot with exon 5a- and 5b-specific probes revealed that the 1.05-and 2.0-kb bands contained exon 5b, whereas sequences matching exon 5a were found in the 2.8-and 7.3-kb bands in addition to the major 1 3-kb band.
35. V. Campuzano et at., data not shown
36. Three of the cDNAs, which are identical in the portion that has been sequenced so far, extend respectively for 0.5, 1, and 2 kb upstream of exon 1. Their sequence presents numerous divergences from X25 in the part corresponding to exon 1, mostly CpG dinucleotides changed to TG or CA, then the sequences are almost identical in the part corresponding to exons 2 to 4 An additional 1 6-kb cDNA begins with a sequence closely matching exon 5a, even in its untranslated region, with only occasional single-base changes and short insertions or deletions [L Montermini et al., in preparation].
37. Southern blot analysis with a cDNA probe of X25 exons 1 to 5a revealed a prominent 5-kb Eco RI band in genomic DNA that did not correspond to any exon and was absent in yeast artificial chromosome and cosmid DNA from the critical FRDA region Several additional bands, also absent from cloned DNA from the FRDA region, appeared when blots were washed at lower stringency (1 × SSC at room temperature). The primers nF2 (5′-TCCCGCGGCCGGCAGAGTT-3′) and E2R (5′-CCAAAGTTCCAGATTTCCTCA-3′), which can amplify a 173-bp fragment spanning exons 1 and 2 of the X25 cDNA, generated a PCR product of corresponding size from genomic DNA, but not from cloned DNA from the FRDA region, indicating the presence of sequences with high similarity to a processed X25 transcript elsewhere in the genome
38. S F Altschul, W. Gish, W Miller, E W Myers, D. J Lipman, J Mol Biol 215, 403 (1990).
39. Secondary structure prediction was performed with the SSP and NNSSP programs, which are designed to locate secondary structure elements [V V Solovyev and A A. Salamov, CABIOS 10, 661 (1994)] The TMpred program was used to predict putative transmembrane domains [K. Hoffmann and W. Stoffel, Biol Chem Hoppe-Seyler 374, 166 (1993)]. PSORT was used to predict possible protein sorting signals [K. Nakai and M Kanehisa, Proteins Struct Funct Genet 11, 95 (1991)].
40. Secondary structure prediction was performed with the SSP and NNSSP programs, which are designed to locate secondary structure elements [V V Solovyev and A A. Salamov, CABIOS 10, 661 (1994)] The TMpred program was used to predict putative transmembrane domains [K. Hoffmann and W. Stoffel, Biol Chem Hoppe-Seyler 374, 166 (1993)]. PSORT was used to predict possible protein sorting signals [K. Nakai and M Kanehisa, Proteins Struct Funct Genet 11, 95 (1991)].
41. Secondary structure prediction was performed with the SSP and NNSSP programs, which are designed to locate secondary structure elements [V V Solovyev and A A. Salamov, CABIOS 10, 661 (1994)] The TMpred program was used to predict putative transmembrane domains [K. Hoffmann and W. Stoffel, Biol Chem Hoppe-Seyler 374, 166 (1993)]. PSORT was used to predict possible protein sorting signals [K. Nakai and M Kanehisa, Proteins Struct Funct Genet 11, 95 (1991)].
42. The following intronic primers were used to amplify the X25 exons: exon 1 (240 bp), F 5′-AGCACCCAGCGCTGGAGG-3′, R: 5′-CCGCGGCTGTTCCCGG-3′; exon 2 (168 bp), F: 5′-AGTAACGTACTTCTTAACTTTGGC-3′, R: 5′-AGAGGAAGATACCTATCACGTG-3′, exon 3 (227 bp). F 5′-AAAATGGAAGCATTTGGTAATCA-3′, R: 5′-AGTGAACTAAAATTCTTAGAGGG-3′, exon 4 (250 bp), F: 5′-AAGCAATGATGACAAAGTGCTAAC-3′, R. 5′-TGGTCCACAATGTCACATTTCGG-3′; exon 5a(223 bp), F: 5′-CTGAAGGGCTGTGCTGTGGA-3′, R 5′-TGTCCTTACAAACGGGGCT-3′; and exon 5b (224 bp), F: 5′-CCCATGCTCAAGACATACTCC-3′, R: 5′-ACAGTAAGGAAAAAACAAACAGCC-3′ Amplifications for exons 2, 3, 4, 5a, and 5b consisted of 30 cycles with the following parameters. 1 mm at 94°C, 1 min at 55°C, and 1 mm at 72°C. To amplify the highly GC-rich exon 1, we raised the annealing temperature to 68°C and 10% dimethyl sulfoxide was added to the reaction. The search for mutations was conducted with single-strand conformation polymorphism analysis [M. Orita, Y. Suzuki, T. Sekiya, K Hayashi, Genomics 5, 874 (1989)] in 168 FRDA patients, and chemical cleavage [J. A. Saleeba, S. J. Ramus, R J H Cotton, Hum Mutat 1, 63 (1992)] in 16 patients.
43. The following intronic primers were used to amplify the X25 exons: exon 1 (240 bp), F 5′-AGCACCCAGCGCTGGAGG-3′, R: 5′-CCGCGGCTGTTCCCGG-3′; exon 2 (168 bp), F: 5′-AGTAACGTACTTCTTAACTTTGGC-3′, R: 5′-AGAGGAAGATACCTATCACGTG-3′, exon 3 (227 bp). F 5′-AAAATGGAAGCATTTGGTAATCA-3′, R: 5′-AGTGAACTAAAATTCTTAGAGGG-3′, exon 4 (250 bp), F: 5′-AAGCAATGATGACAAAGTGCTAAC-3′, R. 5′-TGGTCCACAATGTCACATTTCGG-3′; exon 5a(223 bp), F: 5′-CTGAAGGGCTGTGCTGTGGA-3′, R 5′-TGTCCTTACAAACGGGGCT-3′; and exon 5b (224 bp), F: 5′-CCCATGCTCAAGACATACTCC-3′, R: 5′-ACAGTAAGGAAAAAACAAACAGCC-3′ Amplifications for exons 2, 3, 4, 5a, and 5b consisted of 30 cycles with the following parameters. 1 mm at 94°C, 1 min at 55°C, and 1 mm at 72°C. To amplify the highly GC-rich exon 1, we raised the annealing temperature to 68°C and 10% dimethyl sulfoxide was added to the reaction. The search for mutations was conducted with single-strand conformation polymorphism analysis [M. Orita, Y. Suzuki, T. Sekiya, K Hayashi, Genomics 5, 874 (1989)] in 168 FRDA patients, and chemical cleavage [J. A. Saleeba, S. J. Ramus, R J H Cotton, Hum Mutat 1, 63 (1992)] in 16 patients.
44. Assuming a FRDA carrier frequency in Italy of 1 out of 120 individuals (2) and a frequency of 1154F of 1 out of 40 FRDA chromosomes in Southern Italians, one individual in 3300 in that population is expected to be a carrier of 1154F Finding such a person in a random sample of 210 individuals can occur with >6% probability.
45. The Perkin-Elmer XL long-PCR reagent kit was used to set up the reactions, and standard conditions were used as suggested by the manufacturer with primers 5200Eco (5′-GGGCTGGCAGATTCCTCCAG-3′) and 5200Not (5′-GTAAGTATCCGCGCCGGGAAC-3′). Amplifications were performed in a Perkin-Elmer 9600 machine and consisted of 20 cycles of the following steps: 94°C for 20 s, 68°C for 8 min, followed by 17 cycles in which the length of the 68°C-step was increased by 15 s per cycle. The generated amplification product is 5 kb from normal chromosomes, and about 7.5 kb from FRDA chromosomes.
46. The primers GAA-F (5′-GGGATTGGTTGCCAGTGCTTAAAAGTTAG-3′) and GAA-R (5′-GATCTAAGGACCATCATGGCCACACTTGCC-3′) flank the GAA repeat and generate a PCR product of 457 + 3n bp (n = number of GAA triplets). With these primers, efficient amplification of normal alleles could be obtained by using the traditional PCR procedure with Taq polymerase after 30 cycles consisting of the following steps- 94°C for 45 s, 68°C for 30 s, and 72°C for 2 min. Enlarged alleles were much less efficiently amplified, particularly when present together with a normal allele, therefore, use of these primers is not indicated for FRDA carrier detection A more efficient amplification of expanded alleles, also in FRDA carriers, could be obtained by using the primers Barn (5′-GGAGGGATCCGTCTGGGCAAAGG-3′) and 2500F (5′-CAATCCAGGACAGTCAGGGCTTT-3′). These primers generated a 1.5-kb normal fragment. Amplification was conducted with the long PCR protocol, in 20 cycles composed of the following steps: 94°C for 20 s, 68°C for 2 min and 30 s, followed by 17 cycles in which the length of the 68°C step was increased by 15 s per cycle
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62. This work is dedicated to the memory of Dr. A. E Harding. Supported by grants from NIH (NS34192, M.P. and P.I.P.); from the Muscular Dystrophy Association (M.P.); from the Association Francaise contre les Myopathies, the Groupement de Recherche et d'Etudes sur les Genomes, CNRS, and INSERM (M.K); from the Italian Telethon (S.C); from the Comision Interministerial de Ciencia y Tecnologia; and from the Generalitat Valenciana (F.P.). V C is a recipient of a fellowship from Ministerio de Educacion y Ciencia, Spam, M D M. is a recipient of a fellowship from the Generalitat Valenciana and from the University of Valencia, L M. is a recipient of a fellowship from the Italian Telethon, M.C is a recipient of a fellowship from the Fondation pour la Recherche Médicale, F D is a recipient of a fellowship from the Association Française contre l'Ataxie de Friedreich (AFAF), E M. and J C are recipients of fellowships from the Generalitat Valenciana, and S.B. is a recipient of a fellowship from the Muscular Dystrophy Association. We wish to thank S Vicaire (IGBMC) and P. Milasseau and C. Crueau (Généthon, Evry) for large-scale sequencing. We thank the Baylor College of Medicine Human Genome Center for access to DNA analysis resources and databases. We are grateful to the many clinicians who referred their patients to us, and to all patients and their families for their willingness to participate in this research and for their continuous encouragement.